Salt-tolerant propylene oxide-degrading bacterial strain, culture method and application thereof

Abstract

The present application relates to a kind of salt-tolerant propylene oxide degradation strain and its culture method and application, it solves the technical problems that current strain high salt biochemical system processing efficiency is not high, salt fluctuation and load impact resistance ability is insufficient, high salt microorganism competitive advantage is weak, the strain is named as Exiguobacterium sp. YN-4, its preservation organization is China Microbial Culture Collection Center, and the number of preservation number is: CGMCC No.27797.The present application can be used in the field of propylene oxide production wastewater treatment.

Description

Technical Field

[0001] This invention relates to a strain for use in the environmental protection field, its cultivation method and application, and more specifically, to a salt-tolerant propylene oxide-degrading strain, its cultivation method and application. Background Technology

[0002] Propylene oxide (PO) is an important basic organic chemical raw material. The chlorohydrin process is currently the main method for propylene oxide production both domestically and internationally. Its wastewater is characterized by high temperature, high suspended solids (SS), high pH (11-12), and high calcium salt content (Ca). 2+ (15000 mg / L), high COD Cr Characterized by concentrations of 800–2000 mg / L, and containing large amounts of recalcitrant organic chlorides. The excessively high salt concentration in this type of wastewater inhibits microbial growth, making biological treatment difficult; the wastewater also contains high concentrations of calcium. 2+ When propylene oxide wastewater is aerated and reacts with CO2 in the air, it produces a large amount of CaCO3 precipitate. These precipitates gradually form during the aeration process of biological treatment. These precipitates adhere to the surface of the activated sludge, reducing its activity and significantly decreasing the effectiveness of the biological treatment. Therefore, traditional biological methods struggle to achieve efficient treatment of propylene oxide wastewater.

[0003] Currently, biological methods remain the main treatment for propylene oxide production wastewater, primarily using aerobic processes. However, the high salt content and the biotoxicity of organic matter significantly inhibit the activity and metabolic capacity of conventional microorganisms. Most companies improve the high-salt biochemical treatment effect of propylene oxide wastewater by methods such as salinity acclimatization or the addition of bacterial agents.

[0004] However, existing engineering cases in China show that high-salt biochemical systems have low treatment efficiency, low load and unstable operation, insufficient resistance to salinity fluctuations and load shocks, weak competitive advantage of high-salt microorganisms, and difficulty in maintaining a long-term, stable and efficient treatment level. Summary of the Invention

[0005] This invention aims to address the technical problems of low efficiency in high-salt biochemical systems, insufficient resistance to salinity fluctuations and load shocks, and weak competitive advantage of high-salt microorganisms in existing strains. It provides a salt-tolerant propylene oxide degrading strain that can withstand high salt and has high efficiency in degrading propylene oxide, along with its cultivation method and application.

[0006] Therefore, the present invention provides a salt-tolerant propylene oxide-degrading strain, named Exiguobacterium sp. YN-4, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27797.

[0007] This invention also provides a method for culturing a salt-tolerant propylene oxide-degrading strain, which uses seeds and a fermentation medium with the following concentrations: glycerol 10.0–20.0 g / L, peptone 5.0–10.0 g / L, NaCl 20.0–36.0 g / L, KH₂PO₄ 1.0–1.5 g / L, and MgSO₄·7H₂O 0.5–1.0 g / L.

[0008] Preferably, a microbial protectant is used in the culture, wherein the microbial protectant formulation is 10-20 g / L of polyvinylpyrrolidone (PVP) and 1-3 g / L of xanthan gum.

[0009] This invention also provides the application of a salt-tolerant propylene oxide-degrading strain in the treatment of propylene oxide production wastewater.

[0010] Preferably, a nutrient agent is added during the processing, and the formula of the nutrient agent is: 150-200 g / L soluble starch, 0.01-0.05 g / L yeast extract, 0.4-0.5 g / L ammonium sulfate, and 0.1-0.15 g / L dipotassium hydrogen phosphate.

[0011] The present invention has the following beneficial effects:

[0012] This invention provides a degrading bacterial strain that can grow under high-salt (CaCl2) conditions and remove propylene oxide. It can efficiently degrade more than 80% of propylene oxide in an initial pH range of 7-9 and a salinity range of 1-3%. Attached Figure Description

[0013] Figure 1 This is a morphological image of Exiguobacterium sp. YN-4 provided by the present invention;

[0014] Figure 2 This is a schematic diagram illustrating the effect of temperature on the growth of the bacterial strain in this invention;

[0015] Figure 3 This is a schematic diagram of the growth curve determination of strain YN-4 in Example 3 of the present invention;

[0016] Figure 4 This is a schematic diagram illustrating the effect of salt concentration on the degradation effect of strain YN-4 in Example 4 of the present invention;

[0017] Figure 5 This is a schematic diagram illustrating the effect of pH on the degradation effect of strain YN-4 in Example 4 of the present invention;

[0018] Figure 6 This is a schematic diagram of the degradation of actual wastewater by the bacterial agent YN-4 in Example 5 of the present invention.

[0019] The strain provided by this invention is named Exiguobacterium sp. YN-4, and its depository institution is the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; the accession number is CGMCC No. 27797, and the deposit date is July 5, 2023. Detailed Implementation

[0020] The present invention will be further described below with reference to embodiments.

[0021] The morphological characteristics of the *Exiguobacterium* sp. YN-4 strain provided by this invention in high-salt LB medium are as follows: colonies are pale yellow with round or irregular edges, and the surface is raised, moist, and smooth. They are Gram-positive, non-spore-forming, short rod-shaped cells, occurring singly or in pairs. This strain can treat propylene oxide production wastewater under high-salt conditions.

[0022] Example 1: Enrichment, Screening, and Purification of Propylene Oxide-Degrading Bacterial Strains

[0023] A mixture of activated sludge and sludge from an aerobic tank in a propylene oxide production wastewater facility in Shandong Province was selected and enriched, screened, and separated using the following methods.

[0024] (1) Culture medium formulation

[0025] Enrichment medium (g / L): potassium nitrate 1.0, NaCl 36, MgSO4·7H2O 0.5, FeSO4 0.01, adjust pH to 7.2. Sterilize at 121℃ for 20 min. Add 100 mg / L of propylene oxide to the liquid enrichment medium.

[0026] High-salt LB medium (g / L): 10g peptone, 5g yeast extract, 36g NaCl, with the addition of 2% agar, can be prepared into a solid medium. Adjust the pH to 7.2 and sterilize at 121℃ for 20 min.

[0027] (2) Enrichment and domestication culture

[0028] The collected activated sludge samples were inoculated into 250 mL of sterilized enrichment medium at a 3% inoculum rate; cultured on a shaker at 30°C and 200 rpm for 48 hours; after that, inoculated into fresh enrichment medium at a 3% volume rate; and cultured continuously for 3–5 cycles following the above steps. Finally, the COD degradation rate was quantitatively determined using a Lianhua Technology rapid detection instrument (reagents). Enrichment solutions with significant effects were selected for separation and screening culture.

[0029] (3) Screening, isolation and validation of strains

[0030] 1 mL of the enriched solution, after acclimatization and cultivation, was serially diluted and evenly spread onto solid high-salt LB medium. The cultures were incubated at 30°C for 24–48 h to obtain single colonies of varying sizes and morphologies. Single colonies were then purified using high-salt LB medium. The purified single colonies were inoculated into propylene oxide-containing medium and incubated at 30°C and 200 rpm for 48 h. Degradation capacity was assessed at 24 h and 48 h. The strain with the strongest degradation capacity was selected for preservation, and its characteristics were studied.

[0031] In this embodiment, the above method was used to obtain a degrading bacterial strain, designated YN-4, from more than 10 strains of bacteria. This strain can grow under high-salt conditions and can remove propylene oxide.

[0032] Example 2: Molecular biological identification of strain YN-4

[0033] Homology alignment of the 16S rRNA gene sequence of strain YN-4 and phylogenetic analysis revealed the highest similarity to *Exiguobacterium enclense* NIO-1109 (Accession number: JF893462, 99.43%) and *Exiguobacterium indicum* HHS31 (Accession number: AJ846291, 99.39%), both belonging to the genus *Exiguobacterium*. The strain was named *Exiguobacterium sp.* YN-4 (short for strain YN-4). *Exiguobacterium* spp. are a group of bacteria with a wide distribution and various unique properties, including thermophilicity, psychrophilicity, alkaliphilicity, and halophilicity.

[0034] Morphological characteristics of strain YN-4 in high-salt LB medium after 24 h of culture ( Figure 1 The colonies are pale yellow, with round or irregular edges, and a raised, moist, and smooth surface. They are Gram-positive, non-spore-forming, short rods, occurring singly or in pairs.

[0035] Example 3: Cultivation of strain YN-4

[0036] 1. Effect of temperature on the growth of strain YN-4

[0037] Temperature is a crucial factor affecting microbial growth. In this invention, the growth performance of strain YN-4 was investigated using high-salt LB medium with an initial pH of 7 after culturing at different temperatures (25℃, 30℃, 37℃, and 42℃) for 48 hours. The results showed that strain YN-4 exhibited similar growth at 37℃ and 30℃, followed by 25℃, and was relatively weaker at 42℃. Figure 2 ).

[0038] 2. Growth curve determination of strain YN-4: (e.g.) Figure 3 As shown.

[0039] 3. Preparation of inoculum agent of strain YN-4

[0040] The strain YN-4 was cultured in high-salt LB shake flasks, seed culture, and fermenter culture in sequence.

[0041] Single colonies were picked from high-salt LB solid medium and inoculated into liquid high-salt LB medium. The cultures were then incubated on a shaker at 200 rpm and 30°C for 16 hours. A 3% inoculum was transferred to a seed fermenter containing fermentation medium and incubated at 30°C for 16 hours to obtain the seed culture. A 3% inoculum of the seed culture was then transferred to a fermenter containing fermentation medium. The control parameters for the seed tank and fermenter were: temperature 30°C, aeration rate 1.5 vvm, and rotation speed controlled to approximately 30% dissolved oxygen. After approximately 20 hours of incubation, the OD of the fermentation broth reached 8–10 (viable cell count reached 4.0–5.0 × 10⁻⁶). 9 Fermentation was terminated by adding CFU / mL of the solution. Before transferring the solution to the tank, the pH was adjusted to 6.0. A protective agent was added and the mixture was stirred until homogeneous to obtain strain YN-4.

[0042] The seed and fermentation medium formulation 3a is as follows: glycerol 10.0 g / L, peptone 5.0 g / L, NaCl 20.0 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.5 g / L, pH 7.2; sterilized at 121℃ for 30 min.

[0043] The seed and fermentation medium formulation 3b is as follows: glycerol 20.0 g / L, peptone 10.0 g / L, NaCl 36.0 g / L, KH2PO4 1.5 g / L, MgSO4·7H2O 1.0 g / L.

[0044] The seed and fermentation medium formula 3c is as follows: glycerol 15g / L, peptone 8g / L, NaCl 30g / L, KH2PO4 1.2g / L, MgSO4·7H2O 0.8g / L.

[0045] The 3C formula for seeds and fermentation medium yields the best results.

[0046] The formula for the protectant in this fermentation agent is as follows:

[0047] 3A: Polyvinylpyrrolidone (PVP) 10g / L, xanthan gum 1g / L; 3B: Polyvinylpyrrolidone (PVP) 20g / L, xanthan gum 3g / L; or 3C: Polyvinylpyrrolidone (PVP) 15g / L, xanthan gum 2g / L. Add after the strain has been cultured for 20 hours, and the pH is adjusted to 6.0 before adding to the tank. Formula 3C is the most effective protectant.

[0048] Example 4: Effects of key factors on the degradation efficiency of propylene oxide wastewater by strain YN-4

[0049] 1. Effect of salt concentration on the degradation efficiency of strain YN-4

[0050] The actual wastewater from typical propylene oxide production has a salinity of 1-3%, and is particularly high in calcium (15 g / L). An experiment was designed to investigate the effect of different CaCl2 concentrations on the degradation of propylene oxide by strain YN-4. Fresh bacterial culture was inoculated into shake flasks containing enrichment medium (500 mg / L propylene oxide), with CaCl2 concentrations of 1%, 3%, and 5%. The culture was incubated at 30°C and 180 rpm for 24 and 48 hours, and samples were taken to measure bacterial growth and the epichlorohydrin degradation rate.

[0051] like Figure 4 As shown, the results indicate that the degradation rates of propylene oxide by strain YN-4 at CaCl2 concentrations of 1%, 3%, and 5% were 83.4%, 76.2%, and 43.3%, respectively. The degradation rate decreased with increasing calcium ion concentration, possibly due to salt ion inhibition or the adsorption of insoluble calcium ions onto the bacterial surface, affecting its activity.

[0052] 2. Effect of pH on the degradation efficiency of strain YN-4

[0053] The actual wastewater from typical propylene oxide production is characterized by a high pH value (11-12). The effect of initial pH on the degradation of propylene oxide by strain YN-4 was investigated.

[0054] In an experiment designed to influence the effect of different pH values ​​on the degradation of propylene oxide by strain YN-4, fresh bacterial culture was inoculated into shake flasks containing enrichment medium (containing 500 mg / L of propylene oxide), with pH values ​​set at 7, 8, 9, 10, and 11 respectively. The culture was incubated at 30°C and 180 rpm / min, and samples were taken at 24 h and 48 h to measure the degradation rate of propylene oxide.

[0055] like Figure 5 As shown, the results indicate that the initial pH range of 7 to 9 has little effect on the degradation of propylene oxide by strain YN-4. It was also found that the pH tends to decrease during the degradation of propylene oxide. Therefore, in actual wastewater treatment, the pH can be adjusted to between 8 and 9 to reduce acid consumption during the treatment process.

[0056] Example 5: Application of bacterial agent YN-4 in the degradation of actual wastewater

[0057] The wastewater from a propylene oxide production plant in Shandong Province was selected. The wastewater was mainly composed of propylene oxide, followed by epichlorohydrin, with a pH of 11, COD of 800-1000 mg / L, chloride ion concentration of 21000-22500 mg / L, and ammonia nitrogen ≤20 mg / L.

[0058] A simulated device containing actual wastewater was added. The device employed microporous aeration and was filled with 2 / 3 mesh-modified polyurethane resin packing. To conserve acid, the pH of the raw water was adjusted to approximately 8.5 using sulfuric acid. The control group consisted of pH-adjusted wastewater without added bacteria. Treatment 1 consisted of pH-adjusted wastewater inoculated with 10% by volume of the fermentation agent from Example 4. Treatment 2 consisted of treatment 1 with added nutrients to improve the nutrient balance of the wastewater. Samples were centrifuged, and the supernatant was collected. COD residues were measured every 24 hours.

[0059] Nutrient formula 5a: 15 g / L soluble starch, 0.01 g / L yeast extract, 0.4 g / L ammonium sulfate, and 0.1 g / L dipotassium hydrogen phosphate.

[0060] Nutrient formula 5b: soluble starch 200g / L, yeast extract 0.05g / L, ammonium sulfate 0.5g / L, dipotassium hydrogen phosphate 0.15g / L.

[0061] Nutrient formula 5c: 180g / L soluble starch, 0.04g / L yeast extract, 0.45g / L ammonium sulfate, and 0.12g / L dipotassium hydrogen phosphate.

[0062] The nutrient formula with 5C is the most effective.

[0063] like Figure 6 As shown, the operational results indicate that adding bacteria can effectively degrade COD in wastewater, achieving a degradation rate of 83.4%. Simultaneous use of both microbial agents and nutrients further enhances COD degradation, achieving a degradation rate exceeding 90%. Nutrients promote COD degradation, potentially providing nutrients for microbial growth and a substrate for the co-metabolization of recalcitrant compounds. The results also demonstrate that single microorganisms are insufficient to treat complex, combined wastewater and meet discharge standards.

[0064] However, the above description is merely a specific embodiment of the present invention and should not be construed as limiting the scope of the present invention. Therefore, any substitution of equivalent components or equivalent changes and modifications made in accordance with the scope of protection of the present invention should still fall within the scope of the claims of the present invention.

Claims

1. The application of a salt-tolerant propylene oxide-degrading strain in the treatment of propylene oxide production wastewater, characterized in that, The strain was named Microbacterium ( Exiguobacterium sp. YN-4, its depository institution is the China General Microbiological Culture Collection Center, and its accession number is: CGMCC No.27797.

2. The application of the salt-tolerant propylene oxide-degrading strain according to claim 1 in the treatment of propylene oxide production wastewater, characterized in that, Nutrients need to be added during the processing. The formula of the nutrient is as follows: 150-200 g / L soluble starch, 0.01-0.05 g / L yeast extract, 0.4-0.5 g / L ammonium sulfate, and 0.1-0.15 g / L dipotassium hydrogen phosphate.

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